Many and perhaps most genes of eukaryotes are coded in the genome by multiple noncontinuous sequence blocks separated from one another by intervening sequences. Expression of these genes involves continuous transcription of coding and intervening sequences followed by the splicing of coding sequences to form a mature mRNA. We propose to study the biochemistry of mRNA processing using HeLa cells infected with adenovirus 2 and nondefective Ad2-SV40 hybrid viruses as a model system. SV40 specific mRNAs isolated from cells infected with the Ad2 plus ND viruses are an unexpectedly complex mixture of distinct species in which adenovirus RNA sequences are spliced to SV40 sequences in various ways. We will determine the fine structure of these RNA species using the Berk-Sharp technique and by sequencing the Ad2-SV40 joints. This will provide sequence information regarding the immediate sequences surrounding a large number of splicing sites. Another project will be to characterize the cytoplasmic processing of two Ad2 RNA species in Ad2 infected cells by determining the sequences removed from these RNAs during processing and examining the effect of translation on their ability to be processed. In addition, two activities which can process RNA in vitro have been detected in rabbit reticulocyte lysates. One of these is a site specific endonuclease and the other has apparent RNA splicing activity. These enzymes will be purified and characterized.